Inhibition of DNA methyltransferase

ABSTRACT

The present invention relates to the interplay between the level of DNA methyltransferase and demethylase activities, to the role of the interplay between these levels on the proliferative, differentiated, tumorigenic and homeostatic state of the cell, and to the DNA methyltransferase and demethylase as therapeutic targets. The invention further relates to a reduction of the level of DNA methylation through inhibitors and antagonist in order to inhibit the excessive activity or hypermethylation of DNA MeTase in cancer cells to induce the original cellular tumor suppressing program, to turn on alternative gene expression programs, to provide therapeutics directed at a nodal point of regulation of genetic information, and to modulate the general level of methylase and demethylase enzymatic activity of a cell to permit specific changes in the methylation pattern of a cell.

TECHNICAL FIELD

The present invention relates to the interplay between the level of DNAmethyltransferase and demethylase activities and to the role of thisinterplay on the proliferative, differentiated, tumorigenic andhomeostatic state of the cell.

BACKGROUND ART

While transcription factors play a critical role in orchestrating thegene expression profiles of all organisms, other, “epigenetic” levels ofinformation that encode the diversification program of an otherwiseuniform. genetic content exist. Methylation of DNA is thought to be onesuch critical determinant of the diversification program (Razin et al.,1980, Science 210:604-610).

DNA methylation is a postreplicative covalent modification of DNA thatis catalyzed by the DNA methyltransferase enzyme (MeTase) (Koomar etal., 1994, Nucl. Acids Res. 22:1-10; and Bestor et al., 1988, J. Mol.Biol. 203:971-983). In vertebrates, the cytosine moiety at a fraction ofthe CpG sequences is methylated (60-80%) in a nonrandom mannergenerating a pattern of methylation that is gene and tissue specific(Yisraeli and M. Szyf, 1985, In DNA methylation: Biochemistry andBiological significance, pp. 353-378, Razin et al., (Ed),Springer-Verlag, N.Y.). It is generally believed that methylation inregulatory regions of a gene is correlated with a repressed state of thegene (Yisraeli and Szyf, 1985, In DNA methylation: Biochemistry andBiological significance, pp. 353-378, Razin et al., (Ed),Springer-Verlag, N.Y.; and Razin et al., 1991, Microbiol. Rev.55:451-458). Recent data suggest that DNA methylation can repress geneexpression directly, by inhibiting binding of transcription factors toregulatory sequences or indirectly, by signaling the binding ofmethylated-DNA binding factors that direct repression of gene activity(Razin et al., 1991, Microbiol. Rev. 55:451-458). It is well establishedthat regulated changes in the pattern of DNA methylation occur duringdevelopment and cellular differentiation (Razin et al., 1991, Microbiol.Rev. 55:451-458; and Brandeis et al., 1993, Bioessays 13:709-713).Importantly, the critical role of DNA methylation in differentiation hasrecently been demonstrated (Li et al., 1992, Cell 69:915-926; and Szyfet al., 1992, J. Biol. Chem. 267:12831-12836). The pattern ofmethylation is maintained by the DNA MeTase at the time of replicationand the level of DNA MeTase activity and gene expression is regulatedwith the growth state of different primary (Szyf et al., 1985, J. Biol.Chem. 260:8653-8656) and immortal cell lines (Szyf et al., 1991, J. Bol.Chem. 266:10027-10030). This regulated expression of DNA MeTase has beensuggested to be critical for preserving the pattern of methylation.

Many lines of evidence have demonstrated aberrations in the pattern ofmethylation in transformed cells. For example, the 5′ region of theretinoblastoma (Rb) and Wilms Tumor (WT) genes is methylated in a subsetof tumors, and it has been suggested that inactivation of these genes inthe respective tumors resulted from methylation rather than a mutation.In addition, the short arm of chromosome 11 in certain neoplastic cellsis regionally hypermethylated. Several tumor suppressor genes arethought to be clustered in that area. If the level of DNA MeTaseactivity is critical for maintaining the pattern of methylation as hasbeen suggested before (Szyf, 1991, Biochem. Cell Biol. 64:764-769), onepossible explanation for this observed hypermethylation is the fact thatDNA MeTase is dramatically induced in many tumor cells well beyond thechange in the rate of DNA synthesis. The fact that the DNA MeTasepromoter is activated by the Ras-AP-l signalling pathway is consistentwith the hypothesis that elevation of DNA MeTase activity and resultinghypermethylation in cancer is an effect of activation of the Ras-Junsignalling pathway.

It is clear that the pattern of methylation is established duringdevelopment by sequential de novo methylation and demethylation events(Razin et al., 1991, Microbiol. Rev. 55:451-458; and Brandeis et al.,1993, Bioessays 13:709-713), the pattern being maintained in somaticcells. It is still unclear however, how methylation patterns are formedand maintained In vivo. Although a simple model has been proposed toexplain the clonal inheritance of methylation patterns (Razin et al.,1980, Science 210:604-610), it does not explain how specific sites arede novo methylated or demethylated during the processes ofdifferentiation and cellular transformation. Several lines of evidencesuggest that factors, other than the state of methylation of theparental strand, are involved in targeting specific sites formethylation.

A similar mystery is how specific sites are demethylated duringdevelopment and cellular transformation. One possible mechanism could bea passive loss of methylation, although an alternative hypothesis isthat demethylation is accomplished by an independent enzymaticmachinery.

Site specific loss of methylation is a well documented facet ofvertebrate differentiation (Yisraeli and Szyf, 1985, In DNA methylation:Biochemistry and Biological significance, pp. 353-378, Razin et al.,(Ed), Springer-Verlag, N.Y.; Razin et al., 1991, Microbiol. Rev.55:451-458; and Brandeis et al., 1993, Bioessays 13:709-713). Whereas aloss of methylation could be accomplished by a passive process asdescribed above, a series of observations have demonstrated that anactive process of demethylation occurs in mammalian cells (see forexample Yisraeli et al., 1986, Cell 46:409-416). Similar to de novomethylation, demethylation is directed by specific signals in the DNAsequence (Yisraeli et al., 1986, Cell 46:409-416; and see FIG. 1 hereinfor a model) and the probability of a site being methylated ordemethylated is determined by the affinity of that site to either one ofthe DNA MeTase or demethylase. The affinity of each site to eitherenzyme is determined by the chromatin structure around the site (Szyf,1991, Biochem. Cell Biol. 64:764-769).

In normal cells, the DNA methyltransferase is regulated and repressed,possibly by one of the tumor suppressors. An equilibrium between DNAmethyl-transferase and demethylase activities maintains the methylationpattern. Methylated sites are indicated by (M) in FIG. 1. Inhibition ofthe repressor results in over-expression of the DNA MeTase (as indicatedby the solid arrow) the genome becomes hypermethylated and tumorigenesisis initiated (tumor a). Another mechanism for up regulating the DNAmethyltransferase is the activation of the Ras oncogenic pathwayresulting in activation of Jun and over-expression of the DNA MeTase.However, it appears that the Ras pathway can activate the demethylase aswell. The final pattern of methylation observed in this class of tumorswill reflect both activities: hypermethylation (M) of sites that expresslow or medium affinity to the demethylase (sites 3,4,5) andhypomethylation of sites that are of high affinity but were methylatedin the original cell (site number 6).

The lines of evidence that link cancer and hypermethylation are howeverstill circumstantial. The critical question that remains to be answeredis whether these changes in DNA methylation play a causal role incarcinogenesis.

The demonstration that hypermethylation correlates with carcinogenesiswould be immensely useful since it could lead to methods of assessingthe carcinogenic potential of cells as well as to therapeutic treatmentsof cancer patients. Of note, the fact that the level of DNA MeTase islimiting in mammalian cells is supported by the observation that a smallelevation of cellular DNA MeTase levels by forced expression of anexogenously introduced DNA methyltransferase into NIH 3T3 cells resultsin a significant change in the methylation pattern (Wu et al., 1994,Proc. Natl. Acad Sci. USA 90:8891-8895).

In addition, if DNA methylation provides an important control over thestate of differentiation of mammalian cells, then DNA methylationmodifiers could serve as important therapeutic agents to alter thegenetic program in a predictable manner and/or to restore an authenticprogram when it is disrupted by deregulation of DNA methylation.

Furthermore, the identification of the molecule responsible for thedemethylase activity would be extremely useful for the same reasons asmentioned above, since the control of gene expression, ofdifferentiation and cellular homeostasis appears dependent on thebalance between the level of DNA MeTase and demethylase activities.

DISCLOSURE OF THE INVENTION

The present invention relates to the interplay between the level of DNAmethyltransferase and demethylase activities and to the role of thisinterplay on the proliferative, differentiated, tumorigenic andhomeostatic state of the cell. It relates also to the use of a reductionof a level of methylated cytosine in a CpG dinucleotide, for reversing atransformed state of a cell, for correcting an aberrant methylationpattern in DNA of a cell, or for changing a methylation pattern in DNAof a cell. DNA methyltransferase (DNA MeTase) inhibitors can, accordingto the present invention, be used to inhibit the excessive activity orhypermethylation of DNA MeTase in cancer cells and induce the originalcellular tumor suppressing program. These inhibitors can also be used toturn on alternative gene expression programs. Specific DNAmethyl-transferase antagonists can also provide therapeutics directed ata nodal point of regulation of genetic information. Moreover, thepresent invention relates to the pharmacological implications providedby the fact that specific changes in the methylation pattern of a cellcan be obtained by modulating the general level of DNA MeTase anddemethylase enzymatic activity of that cell. Therefore, silent genes canbe activated through a change in the methylation pattern of the DNA. Forexample, β-thalassemia and sickle cell anemia can be treated byactivating the β-globin gene following a change in its methylationpattern.

Based on the demonstration that over expression of DNA MeTase in NIH 3T3cells resulted in cellular transformation (Wu et al., 1994, Proc. Natl.Acad Sci. USA 90:8891-8895), the present invention also relates to theDNA MeTase as a candidate target for anticancer therapy.

The present invention also relates to a recently purified demethylaseactivity from P19 cells and to the demonstration that this demethylaseis induced in P19 cells transformed with RAS. Based on the increasedhypomethylation in cancer, and the demonstration that the demethylasefrom P19 cells is induced by RAS, the present invention further relatesto the demethylase as a candidate target for anticancer therapy.Furthermore, the demethylase activity could be extremely useful for thetreatment of methylated DNA samples that are to be used in molecularanalysis such as restriction mapping or cloning.

The present invention moreover relates to poly clonal or monoclonalantibodies directed against the DNA MeTase or demethylase, and to theuse of such antibodies as therapeutic agents.

Moreover, the present invention relates to the use of DNA MeTaseinhibitors, whether general or specific, as anticancer therapeuticagents.

In a preferred embodiment, the specific anticancer therapeutic agent isan antisense oligonucleotide, specific to DNA MeTase mRNA sequences. Ina case wherein hypermethylation of tumor suppressor loci results intheir repression, and demethylation results in the activation of genesencoding tumor stimulators thereby amounting to the induction oftumorigenesis, initiating antisense therapy against the DNAmethyltransferase will result in a reduction in DNA MeTase activity,demethylation and reactivation of tumor suppressor genes. The productsof these genes will inhibit the tumorigenic effect induced by the tumorstimulating genes and thus, the inhibition of hypermethylation will alsoinhibit the effects of hypomethylation.

In an other preferred embodiment, based on the crystal structure of theHhaI methylase (Kumar et al., 1994, Nucl. Acids Res. 22:1-10),presenting a detailed atomic structure of the DNA methyltransferase, itis now possible to rationally design highly specific antagonists. Thesenovel antagonists can be potential candidates for anticancer and geneinduction therapy. The potential advantage of anti DNA MeTase therapyover alternative chemotherapy approaches is that it targets a potentialregulator of the cancer state rather than a nonspecific proliferativefunction. DNA MeTase inhibitors can thus provide a novel route oftherapy directed at the regulation of the genetic information.

In yet another preferred embodiment, through the use of antisensetherapy, reversal of the tumorigenic phenotype of the cell can beobserved.

In the specification and appended claims the antisense designationshould be interpreted as being a DNA or RNA molecule complementary tothe MRNA or towards either of the two DNA strands against which it istargeted. This antisense can be a complementary full length version ofthe target sequence, a fragment thereof, or an oligonucleotide derivedtherefrom. This antisense can be obtained by biotechnological methods orsynthesized chemically.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that DNA MeTase and demethylase activities determinethe pattern of DNA methylation in tumor cells;

FIG. 2A illustrates plasmids pZEM and pZαM. The metallothionine (MT)promoter (shaded box), the human growth hormone 3′ region (HGH) (openbar), and the MeTase cDNA sequences (hatched) are indicated;

FIG. 2B shows a Southern blot analysis verifying the presence of thetransfected plasmid in the transfectants;

FIG. 2C shows a Northern blot analysis of the positive clones expressingthe expected 1.3 kb chimeric mRNA. Total RNA (5 μg) was prepared fromthe three pZαM lines (7 and 9) and from the pZEM;

FIGS. 3A, 3B show the state of methylation of total genomic DNA inYlpZαM transfectants by nearest neighbour analysis of 2 ug DNA extractedfrom pZαM transfectant (4) and a pZEM control. FIG. 3A shows anautoradiogram of a representative TLC plate. The standard lane is ofhemimethylated M13 DNA synthesized In vitro. FIG. 3B shows scintillationcounts from spots corresponding to C and 5-methyl C. The valuesrepresent the means ± SEM;

FIGS. 3C-3F. show Southern blot analyses following MspI/HpaII digestion(M/H) of the DNA illustrating, in YlpZαM transfectants, the state ofmethylation of specific genes: 3C: the C21 5′ region, 3D: the C21 gene,3E: the retinoblastoma (RB) gene and 3F: the p53 gene. In FIGS. 3C-3E,the open arrows indicate the position of demethylated fragments.

FIGS. 4A-4C shows the morphological transformation and reduced anchorageindependent growth of Yl cells transfected with pZαM. FIG. 4A shows aPhase contrast microscopy at ×200 magnification of living cultures of Ylclonal transfectants with pZαM and Yl controls. FIG. 4B shows picturesof phase contrast microscopy at ×10 of a 21 days soft agar assay of YlpZEM cells (clones 4 and 7) and Yl pzαM transfectants (clones 4, 7 and9). FIG. 4C shows an anchorage independent growth assay: Yl pZEM (clones4 and 7) and Yl pZαM transfectants (clones 4, 7 and 9) after 21 days ofgrowth in soft agar;

FIG. 5A-5B show the In vivo tumorigenicity of YlpZαM transfectants. FIG.5A illustrates the ability of two control lines (Yl and pZEM4) and threeYlpZαM transfectants (4, 7 and 9) to form tumors in LAF-l mice, as wellas the level of neovascularization in these tumors. FIG. 5B showsphotographs of the homogenized tumors;

FIGS. 6A-6B show the loss of antisense expression in tumors derived fromYlpZαM transfectants by Northern blot analysis. The 1.3 Kb antisensemessage is seen only in the original cell line pZαM 7 (dark arrow), andis undetectable in tumors arising from pZαM 7 or Yl cell lines. Acontrol for the amount of RNA loaded is also shown. FIG. 6B shows therelative expression of the antisense normalized to that of the 18Ssignal;

FIG. 7A shows a density restricted growth assay of Yl pZαM relative tocontrol pZEM transfectants;

FIG. 7B shows the percentage of viable cells as determined using trypanblue staining following serum-deprivation (1% horse serum);

FIG. 7C shows a Southern analysis of total cellular DNA from theindicated transfectants following growth in 1% serum containing mediumand harvested after 1 and 2 days. A 130 bp internucleosomal laddercharacteristic of cells dying via apoptosis can be seen in the YlpZdMtransfectants only;

FIG. 7D shows an electron microscopic analysis of various Yltransfectants cell sections (I-V), following growth of the cells in 1%serum medium for 24 hours.

FIGS. 8A-8D shows the effect of 5 azaCdR-treatment (0-10 μM) of Ylcells. FIG. 8A shows the content of nonmethylated cytosines in thedinucleotide sequence CpG as determined by a nearest neighbour analysis.FIG. 8B shows the effect of 5azaCdR on the viability of cells grown inlow (1%) serum medium. FIG. 8C shows the anchorage independent growth insoft agar (in the absence of 5 azaCdR). FIG. 8D shows the number ofcolonies upon 5azaCdr treatment.

FIG. 9 illustrates the Regulation mechanism of the DNA MeTase promoterwhich determines DNA methylation patterns and cellular transformation.

MODES FOR CARRYING OUT THE INVENTION

It has been previously demonstrated that forced expression of an“antisense” mRNA to the most 5′ 600 bp or the DNA MeTase message (pZαM)can induce limited DNA demethylation in 10 T ½ cells (Szyf et al., 1992,J. Biol. Chem. 267:12831-12836). To directly test the hypothesis thatthe tumorigenicity of Yl cells is controlled by the DNA MeTase, Yl cellswere transfected with either pZαM or a pZEM control.

I. Expression of Antisense to the DNA Methyltransferase Gene in Yl CellsResults in Limited DNA Demethylation.

To directly inhibit DNA methylation in Yl cells, the DNA MeTaseantisense expression construct pZαM or a pZEM control vector (Szyf etal., 1992, J. Biol. Chem. 267:12831-12836) were introduced into Yl cellsby DNA mediated gene transfer. Yl cells were maintained as monolayers inF-10 medium which was supplemented with 7.25% heat inactivated horseserum and 2.5% heat inactivated fetal calf serum (Immunocorp, Montreal).All other media and reagents for cell culture were obtained fromGIBCO-BRL. Yl cells (1×10⁶) were plated on a 150 mm dish (Nunc) 15 hoursbefore transfection. The pZαM expression vector encoding the 5′ of themurine DNA MeTase cDNA (10 ug) was cointroduced into Yl cells with 1 ugof pUCSVneo as a selectable marker by DNA mediated gene transfer usingthe calcium phosphate protocol (Ausubel et al., 1988, In CurrentProtocols in Molecular Biology. Wiley and Sons, New York). Selection wasinitiated 48 hours after transfection by adding 0.25 mg/ml G418(GIBCO-BRL) to the medium. G418 resistant cells were cloned in selectivemedium. For analysis of growth in soft agar, l×10³ cells were seeded intriplicate onto 30 mm dishes (Falcon) with 4 ml of F-10 mediumcontaining 7.5% horse serum, 2.5% FCS, 0.25 mg/ml G418 (fortransfectants) and 0.33% agar solution at 37° C. (Freedman et al.,1974,Cell 3: 355-359). Cells were fed with 2 ml of medium plus G418 every twodays. Growth was scored as colonies containing >10 cells, 21 days afterplating.

G418-resistant colonies were isolated and propagated for bothconstructs. To confirm that the transfectants bear the introducedconstruct, we prepared DNA from the transfectants and subjected it todigestion by either MspI or HpaII, Southern blot analysis andhybridization with a 32p labelled 0.6 kb DNA MeTase cDNA fragment (FIG.2A). Preparation of genomic DNA and all other standard molecular biologymanipulations, such as Labelling (using the random primer labelling kitfrom Boehringer Mannheim), were performed according to Ausubel et al.,1988, In Current Protocols in Molecular Biology. Wiley and Sons, NewYork). MspI and HpaII restriction enzymes (Boehringer Mannheim) wereadded to DNA at a concentration of 2.5 units/ug for 8 h at 37° C.Radionucleotides (3000 mCi/mmol) were purchased from Amersham. Theresults presented in FIG. 2B demonstrate that the three pZαMtransfectants contained significant levels of the DNA MeTase cDNAsequence while the control transfectants were clean. To test whether thepZαM construct is expressed in the transfectants and whether themetallothionein promoter is functional in these cells, we cultured thetransfectants with 50 uM of ZnS04, prepared RNA at different timepoints, subjected it to Northern blot analysis and hybridization withthe 32p labelled MET 0.6 probe. Preparation of total cellular RNA,blotting RNA on to Hybond-N+ (Amersham), were performed according toAusubel et al., (1988, In Current Protocols in Molecular Biology. Wileyand Sons, New York). As observed in FIG. 2C the transfectants 7 and 9express substantial amounts of the MET 0.6 cDNA (˜1.3 kb chimeric MRNA)even before induction with ZnS04. The ZnS04 induction increases therelative intensity of a 1.3 kb RNA hybridizing to MET 0.6 suggestingthat ZnS04 induces the initiation of transcription from a discrete sitebut not the total expression of the antisense message (resulting in asmear in the non induced RNA samples). In subsequent experiments,induction of the transfectants with ZnS04 was therefore not carried out.

To determine whether expression of antisense RNA to the DNA MeTase geneleads to a general reduction in the level of methylation of the genome,we resorted to “nearest neighbour” analysis using [α-32P]-dGTP aspreviously was performed. This assay enables the determination of thepercentage of methylated cytosines residing in the dinucleotide sequenceCpG (Razin et al., 1985, IN Biochemistry and Biology of DNA methylation,p. 239, Razin et al., (Ed), Allan R. Liss, Inc. N.Y.). Briefly, two ugof DNA were incubated at 37° C. for 15 minutes with 0.1 unit of DNAase,2.5 of 32P-α-dGTP (3000 Ci/mmol from Amersham) and 2 units of KornbergDNA polymerase (Boehringer) were then added and the reaction wasincubated for an additional 25 minutes at 30° C. 50 ul of water wereadded and the nonincorporated nucleotides were removed by spinningthrough a microcon™ column (Amicon) at maximum speed for 30 seconds. Thelabelled DNA (20 ul) was digested with 70 ug of micrococal nuclease(Pharmacia) in the manufacturer's recommended buffer for 10 hours at 37°C. Equal amounts of radioactivity were loaded on TLC phosphocelluloseplates (Merck) and the 3′ mononucleotides were separated bychromatography in one dimension (iso-butyric acid: H₂O:NH₄OH in theratio 66:33:1). The chromatograms were exposed to XAR™ film(Eastman-Kodak) and the spots corresponding to cytosine and5-methylcytosine were scraped and counted in a β-scintillation counter.

Transfectants and control DNAs were nicked with DNAaseI, nick translatedwith a single nucleotide [α-32P]-dGTP using DNA polymerase I and thelabelled DNA was digested to 3′ mononucleotide phosphates withmicrococal nuclease which cleaves DNA 3 ′ to the introduced α-32p. The[α-32P] labelled 5′ neighbours of dGMP were separated by chromatographyon a TLC plate, the resulting spots for dCMP and dC^(met)MP were scrapedand counted by liquid scintillation. The results of a triplicateexperiment presented in FIG. 3A (sample autoradiogram) and B (graphicrepresentation) suggest that a limited but significant reduction in thetotal level of DNA methylation (12% for transfectant number 4 and 22%for 7) occurred in transfectants expressing the pZαM construct whencompared to the control line pZEM.

II. Demethylation of Specific Genes in Yl pZαM Transfectants.

To further verify that expression of pZαM results in demethylation andto determine whether specific genes were demethylated, we resorted to aHpaII/MspI restriction enzyme analysis followed by Southern blotting andhybridization with specific gene probes. HpaII cleaves the sequenceCCGG, a subset of the CpG dinucleotide sequences, only when the site isunmethylated while MspI will cleave the same sequence irrespective ofits state of methylation. By comparing the pattern of HpaII cleavage ofspecific genes in cells expressing pZαM with that of the parental Yl orcells harboring only the vector, it can be determined whether the genesare demethylated in the antisense transfectants. The state ofmethylation of the steroid 21-hydroxylase gene C21 (Szyf et al., 1989,Proc. Natl. Acad. Sci. USA 86: 6853-6857; and Szyf et al., 1990,Mol.Endocrinol. 4:1144-1152) was first analyzed. This gene isspecifically expressed and hypomethylated in the adrenal cortex but isinactivated and hypermethylated in Yl cells. It has been previouslysuggested that hypermethylation of C21 in Yl cell is part of thetransformation program that includes the shut down of certaindifferentiated functions. DNA prepared from Yl, pZαm and pZEM (Bernardset al., 1989,. Proc. Natl. Acad. Sci. USA. 86:6474-6478) transfectantswas subjected to either MspI or HpaII digestion, Southern blot analysisand hybridization with a 0.36 kb Xba-BamHI fragment containing theenhancer and promoter regions of the C21 gene (see references Szyf etal., 1989, Proc. Natl. Acad. Sci. USA 86:6853-6857; and Szyf et al.,1990, Mol. Endocrinol. 4:1144-1152, for a physical map of the probe).This probe should detect 0.36 kb and 0.16 kb HpaII fragments when thepromoter region is fully demethylated. The promoter and enhancer regionis heavily methylated in Yl cells and the pZEM transfectants asindicated by the presence of the higher molecular weight partial HpaIIfragments at 3.8 and 2 kb and the absence of any lower molecular weightfragments (FIG. 3C). In contrast, the Yl pZαM transfectants bear apartially demethylated C21 5′ region as indicated by the relativediminution of the 3.8 and 2 kb fragments and the appearance of the fullydemethylated faint bands at 0.36 kb as well as as the fact that HpaIIcleavage yields partial fragments at 0.56 and ˜1 kb indicating partialhypomethylation of sites upstream and downstream to the enhancer region(FIG. 3C). To determine whether hypomethylation was limited to theenhancer region or if it spreads throughout the C21 gene locus, asimilar HpaII digestion and Southern blot transfer on differentpreparations of DNA extracted from Yl cells was performed. DNA from acontrol pZEM (Bernards et al., 1989,. Proc. Natl. Acad. Sci. USA. 86:6474-6478) transfectant and three pZαM antisense transfectants (FIG. 3D)was hybridized to the filter with a 3.8 kb BamHI fragment containing thebody of the C21 gene and 3′ sequences. Full demethylation of this regionshould yield a doublet at ˜1 kb, a 0.8 kb fragment and a 0.4 kb fragmentas well as a number of low molecular weight fragments at 0.1-0.2 kb. Asobserved in FIG. 3D the C21 locus is heavily methylated in Yl cells aswell as the control transfectant as indicated by the high molecularweight fragments above 23 kb. Only a faint band is present in theexpected 1 kb molecular weight range as well as a partial at 1.9 kb(FIG. 3D). The DNA extracted from the antisense transfectants exhibits arelative diminution of the high molecular weight fragments and relativeintensification of the partial fragment at 1.9 kb as well as theappearance of new partial fragments in the lower molecular weight rangebetween 1 and 0.4 kb indicating partial hypomethylation at large numberof HpaII sites contained in the 3′ region of the C21 gene. The patternof demethylation, indicated by the large number of partial HpaIIfragments (FIG. 3D), is compatible with a general partialhypomethylation rather than a specific loss of methylation in a distinctregion of the C21 gene.

To determine whether demethylation is limited to genes that arepotentially expressible in Yl cells such as the adrenal cortex-specificC21 gene or if the demethylation is widely spread in the genome, othergenes such as the muscle specific MyoD gene as well as the hippocampusspecific 5HTlA receptor gene were analysed, and both genes were shown tobe hypomethylated. Another class of genes that might have undergone aspecific hypomethylation includes the tumor suppressor genes. The stateof methylation of two genes from this class, p53 and retinoblastoma (RB)which are both tumor suppressor genes involved in cell cycle regulationwas therefore determined. Loss of either one of these gene products hasbeen shown to lead to deregulation of the cell cycle and neoplasia.

Oligoprimers for the 5′region of the mouse p53 gene were selected fromthe published genomic sequence (Accession number: X01235) using thePrimer selecting program (PC Gene™). The 5′ primer corresponding tobases 154-172: 5′TLC GAA TCG GTT TLC ACCC 3′ SEQ ID NO:1 and the 3′primer corresponding to bases 472-492, 5′ GGA GGA TGA GGG CCT GAA TGC3′SEQ ID NO:2, were added to an amplification reaction mixturecontaining 100 ng of mouse DNA (from C2C12 cells) using the incubationconditions recommended by the manufacturer (Amersham Hot tub™; 1.5 mMMgCl₂) and the DNA was amplified for 40 cycles of 2 minutes at 95° C., 2minutes at 55° C. and 0.5 minutes at 72° C . The reaction products wereseparated on a low-melt agarose gel (BRL) and the band corresponding tothe expected size was excised and extracted according to standardprotocols (Ausubel et al., 1988, In Current Protocols in MolecularBiology, Wiley and Sons, New York).

Since the genomic sequence of the mouse RB gene was unavailable throughGenbank, we reverse transcribed the retinoblastoma mRNA from 0.5 ug oftotal mouse RNA (from C2C12 cells) using random oligonucleotide primers(Boehringer) with Superscript™ reverse transcriptase (BRL) underconditions recommended by the manufacturer. The RB sequence wasamplified from the reverse transcribed cDNA using oligonucleotidescorresponding to bases 2-628 of the published cDNA (Bernards et al.,1989,. Proc. Natl. Acad. Sci. USA. 86:6474-6478). The oligoprimers usedwere 5′ GGA CTG GGG TGA GGA CGG 3′ SEQ ID NO:3 (1-18) and 5′ TTT CAG TAGATA ACG CAC TGC TGG 3′ SEQ ID NO:4 (620-610). The amplificationconditions were as described above.

Using a probe to a 300 bp sequence from the 5′ region of the mouse RBcDNA the level of methylation of this gene in Yl cells transfected witha control vector as well as the pZαM transfectants was determined (FIG.3E). Cleavage of this region with HpaII yields 0.6 kb and 0.1 kbfragments (FIG. 3E). The RB locus is heavily methylated in the controlcells as indicated by hybridization of the probe to high molecularweight fragments. This locus is partially hypomethylated in the pZαMtransfectants as indicated by the relative diminution in the intensityof the high molecular weight fragments, the appearance of numerouspartial fragments between 23 and 0.6 kb and the appearance of thedemethylated fragments at 0.6 kb and ˜0.1 kb.

The p53 locus was studied using a 0.3 kb fragment from the 5′ region 300bp upstream to the initiation site as a probe (FIG. 3F). Cleavage of thep53 loci (two p53 genes are present in the mouse genome) with MspIyields fragments in the 4.4, 2.5, 0,56 and 0.44 kb molecular weightrange (FIG. 3F, first lane). Cleavage of the control Yl pZEMtransfectants shows that only the sites flanking the 0.56 kb fragmentsare demethylated in Yl cells. The rest of the locus is heavilymethylated as indicated by the intensity of the signal at the >4.4 kbrange (FIG. 3F, lanes 2-4). In comparison to the control transfectantsthe p53 gene is partially hypomethylated in Yl cells expressing anantisense message to the DNA MeTase as implied by the relative reductionin the intensity of the high molecular weight fragments above 4.4 kb andappearance of the 4.4 kb HpaII fragment, the partially cleaved HpaIIfragment at 4 kb, the faint partial fragment around 3.5 kb and the faintfragment at 2.5 kb (FIG. 3F last three lanes). These results furthersubstantiate the conclusion that expression of an antisense to the DNAMeTase results in a genome-wide partial hypomethylation. Neither of thegenes studied demonstrates a distinct selectivity in demethylation.

EXAMPLE 1 Morphological Transformation and Loss of Anchorage IndependentGrowth of Yl Cells Expressing Antisense to the DNA MeTase.

To determine whether demethylation induced by the DNA MeTase antisenseconstruct results in a change in the growth properties of cancer cells,the growth and morphological characteristics of the pZαM transfectantsversus the controls we compared. To compare the growth curve of pZαMtransfectants and controls, 5×10⁴ Yl pZEM and pZαM transfectants (4 and7) cells were plated in triplicate. The cells were harvested and countedat the indicated time points (FIG. 7A). The results of this experimentshow that the antisense transfectants reach saturation density at lowerconcentrations than the control cells suggesting that the transfectantshave reacquired “contact inhibition” which is one of the traits lost incancer cells. The morphological properties of the Yl pZαM transfectantsfurther support this conclusion (FIG. 4A). While control Yl and Yl pZEMcells exhibit limited contact inhibition and form multilayer foci, YlpZαM transfectants exhibit a more rounded and distinct morphology andgrow exclusively in monolayers (FIG. 4A).

To determine whether the expression of antisense to the DNA MeTaseresults in reversal of the tumorigenic potential the ability of thetransfectants to grow in an anchorage independent fashion, which isconsidered an indicator of tumorigenicity, was also determined. The YlpZαM transfectants demonstrate an almost complete loss of ability toform colonies in soft agar, moreover the colonies that do form containonly a few cells as demonstrated in FIG. 4B. Growth on soft agar wasquantified by visual examination and presented graphically in FIG. 4C.

These experiments demonstrate that inhibition of DNA methylation byexpression of an antisense message to the DNA MeTase leads to loss oftumorigenicity In vitro.

EXAMPLE 2 Yl Cells Expressing Antisense to the DNA MeTase ExhibitDecreased Tumorigenicity In vivo

To determine whether demethylation can result in inhibition oftumorigenesis In vivo, LAF-l mice (6-8 week old males) were injectedsubcutaneously (in the flank area) with 10⁶ cells for each of the YlpZαM, Yl and Yl pZEM transfectants. Mice were monitored for the presenceof tumors by daily palpitation. Mice bearing tumors of greater than 1 cmin diameter were sacrificed by asphyxiation with CO₂, tumors wereremoved by dissection and homogenized in guanidium isothiocyanate. Micethat were tumor free were kept for ninety days and then sacrificed. RNAwas prepared from the tumors by CsCl₂ density gradient centrifugation asdescribed (Ausubel et al., 1988, In Current Protocols in MolecularBiology, Wiley and Sons, New York). While all the animals injected withYl cells formed tumors two to three weeks post-injection, the rate oftumor formation in the animals injected with the pZαM transfectants wassignificantly lower (FIG. 5A; p>0.005).

Many lines of evidence suggest that angiogenic potential and metastaticpotential of cell lines are directly related. The tumors that do arisefrom the pZαM transfectants exhibit very limited neovascularization(FIG. 5B) while tumors that formed in the animals that were injectedwith Yl cells or control transfectants were highly vascularized (FIG.5B). This difference in neovascularization is indicated by the palecolor of the homogenates of tumors removed from animals injected with YlpZαM transfectants cells versus the very dark homogenates of tumorsarising from control lines (Yl and YlpZEM; FIG. 5B).

One possible explanation for the fact that a small number of tumors didform in animals injected with the pZαM transfectants is that they arederived from revertants that lost expression of the antisense to the DNAMeTase under the selective pressure In vivo. This hypothesis was testedwith isolated RNA from a tumor arising from the YlpZαM transfectant, andcompared to the level of expression of the 0.6 kb antisense messageobserved for the transfectant line In vitro. The isolated RNAs weresubjected to Northern blot analysis and hybridization with a ³²plabelled MET 0.6 fragment. The filter was stripped of its radioactivityand was rehybridized with a ³²P labelled oligonucleotide probe for 18SrRNA (FIG. 6A) as previously described (Szyf et al., 1990, Mol.Endocrinol. 4:1144-1152). The autoradiograms were scanned and the levelof expression of MET 0.6 was determined relative to the signal obtainedwith the 18S probe (FIG. 6B). The expression of the antisense message issignificantly reduced in the tumors supporting the hypothesis thatexpression of an antisense message to the DNA MeTase is incompatiblewith tumorigenesis.

EXAMPLE 3 Expression of pZαM in Yl Cells Leads to an Induction of anApoptotic Death Program upon Serum Deprivation

Tumor cells exhibit limited dependence on serum and are usually capableof serum independent growth. Factors present in the serum are essentialfor the survival of many nontumorigenic cells. Several lines of evidencehave recently suggested that the enhanced survivability of tumorigeniccells is associated with inhibition of programmed cell death. Forexample, the oncogene bc1-2 is not a stimulator of cell proliferationbut rather causes inhibition of apoptosis. The tumor suppressor p53 caninduce apoptosis in a human colon tumor derived line and certainchemotherapeutic agents have been shown to induce apoptosis in cancercells. Since the pZαM transfectants appeared to demonstrate an enhanceddependence on serum and limited survivability under serum deprivedconditions, the possibility that demethylation can induce an apoptoticprogram in Yl cells was analyzed. It was reasoned that as factors in theserum are known to act as survival factors for cells, an apoptoticprogram could be activated only when these factors are remove. To testwhether pZαM transfectants undergo programmed cell death under serumdeprived condition, the effects of serum starvation on thesetransfectants was studied. pZαM transfectants and control Yl pZEMtransfectants (3×10⁵ per well) were plated in low serum medium (1% horseserum) in six well plates, harvested every 24 hours and tested forviability by trypan blue staining (FIG. 7B). While the control cellsexhibited almost 100% viability up to 72 hours after transfer into serumdeprived medium, the Yl pZαM cells showed up to 75% loss of viability at48 hours (FIG. 7B).

The rapid onset of death in Yl pZαM clones under serum deprivedconditions suggests that an active process is involved. Severalobservable changes distinguish apoptosis from necrosis: apoptosis is anactive process requiring de novo protein synthesis; apoptosis isassociated with death of isolated cells, unlike necrosis where patchesor areas of tissue die; cells dying by apoptosis do not elicit an immuneresponse; and the most diagnostic feature of apoptosis is the pattern ofdegradation of the DNA from apoptotic cells (Ellis et al., 1991, Annu.Rev. Cell Biol. 7:663-698). DNA from cells dying by apoptosis generallyexhibit a characteristic ladder when analyzed by gel electrophoresisbecause Ca²⁺/Mg²⁺ dependent endonucleases cleave the DNA atinternucleosomal regions (Ellis et al., 1991, Annu. Rev. Cell Biol.7:663-698). Although the appearance of the 180 bp internucleosomalladder is a diagnostic feature of apoptotic death, other morphologicalchanges such as chromatin condensation, cellular fragmentation; andformation of apoptotic bodies are generally considered to be earlierevents in the apoptotic process and therefore also serve as usefulmarkers. To test whether the serum deprived Yl pZαM cells were dying asa result of an activated apoptotic death program, cells were plated instarvation medium (1% horse serum) and harvested at 24 hour intervals.Total cellular DNA was isolated from the cells and was subjected toelectrophoresis on a 1.5% agarose gel followed by transfer to nylonmembrane and hybridization with random labeled Yl genomic DNA. After 48hours in serum starved conditions, pZαM transfectants exhibit thecharacteristic 180 bp internucleosomal DNA ladder while the control pZEMtransfectants show no apoptosis at this time point (FIG. 7C).

To determine whether cells expressing antisense to the DNA MeTaseexhibit early morphological markers of apoptosis, cells were serumstarved for 24 hours (2% horse serum), harvested and analyzed byelectron microscopy. For electron microscopy, cells were fixed inglutaraldehyde (2.5%) in cacodylate buffer (0.1M) for one hour andfurther fixed in 1% osmium tetroxide. The samples were dehydrated inascending alcohol concentrations and propylene oxide followed byembedding in Epon. Semithin sections (lum) were cut from blocks with anultramicrotome, counterstained with uranil acetate and lead citrate.Samples were analyzed using a Philips 410 electron microscope (Maysingeret al., 1993, Neurochem Intl. 23: 123-129). FIG. 7D shows the electronmicrographs of control Yl pZEM and Yl pZαM transfectants at variousmagnifications (I-V). The control cells have a fine uniform nuclearmembrane whereas the pZαM cells exhibit the cardinal markers ofapoptosis: condensation of chromatin and its margination at the nuclearperiphery (panels I and II), chromatin condensation (panel II), nuclearfragmentation (panel III), formation of apoptotic bodies (panel V) andcellular fragmentation (panel IV). This set of experiments suggests thatone possible mechanism through which demethylation can inhibittumorigenesis is by activating programmed cell death. This is supportedby data suggesting that cell death is triggered by an endonucleaseactivity (Ellis et al., 1991, Annu. Rev. Cell Biol. 7: 663-698). Thus,effects on the DNA methylation levels, can affect the pathway leading toapoptosis.

EXAMPLE 4 Treatment of Yl Cells with 5azaCdR

1×10⁵ yl cells were plated in growth medium. Twenty-four hours afterplating, the medium was replaced with fresh medium containing variousconcentrations (0-10 μM of 5 azaCdR (Sigma). The medium was removed andreplaced with fresh medium containing 5 azaCdR every 12 h for a periodof 72 h. Following 5 azaCdR treatment the cells were plated onto a sixwell dish in growth medium (100, 300, 500 cells per well) forcologinecity determinations, in soft agar for determining anchorageindependent growth (3×10³ cells per well) and in low serum (1% horseserum) for five days to determine viability under serum deprivedconditions. All of these assays were performed in the absence of 5azaCdR.

As shown in FIG. 8, treatment of Yl cells with 5 azaCdR mimics theaction of the expression of an antisense for MeTase. Indeed, treatmentof Yl cells with 5 azaCdR is shown to increase the level ofnon-methylated cytosine (FIG. 8A), to decrease the viability ofserum-starved cells (FIG. 8B), and finally to drastically inhibit thegrowth of Yl cells on soft agar (FIG. 8C-D). The effect of 5 azaCdR onYl cells was shown not to depend on the cell line per se sinceperforming the same experiment under the same conditions but usingRb-human tumors and human small cell lung carcinoma cells gave similarresults.

This set of experiment therefore suggests that 5 azaCdR can besuccessfully used as an anticancer agent, to alter the genetic programor to restore an authentic program disrupted by deregulation of DNAmethylation.

The data presented herein, strongly support the hypothesis thathypermethylation plays a critical role in maintenance of the transformedstate, and even predict that the increase in methylation is critical forthe transformed state. The fact that the RAS signalling pathway has beenshown to induce the activity of the DNA MeTase promoter provides us witha mechanism to explain this increase in the DNA methylation capacity ofcancer cells (FIG. 9). It stands to reason therefore that the DNA MeTaseis an important effector of the RAS signalling pathway.

Taken together, the data presented above, provide basic principlesregarding the therapeutic implications of DNA methylation. First,because the level of DNA MeTase activity is one determinant of thepattern of DNA methylation, partial inhibition of DNA MeTase activitycan result in a change in the methylation pattern. If aberranthypermethylation in cancer cells is caused by over-expression of the DNAmethyltransferase, then partial inhibition of methylation is expected torestore the original methylation pattern. Second, since this pattern isnot exclusively determined by the DNA MeTase activity but is alsodefined by cis- and trans-acting signals at the gene site, a partialinhibition of DNA MeTase will result in a programmed change in geneexpression rather than a chaotic transformation of the cell.Furthermore, DNA MeTase inhibitors can be used to induce a program thatis latent in the cell.

While the invention has been described with particular reference to theillustrated embodiment, it will be understood that numerousmodifications thereto will appear to those skilled in the art.Accordingly, the above description and accompanying drawings should betaken as illustrative of the invention and not in a limiting sense.

4 19 base pairs nucleic acid single linear DNA NO NO unknown 1TCCGAATCGG TTTCCACCC 19 21 base pairs nucleic acid single linear DNA NONO unknown 2 GGAGGATGAG GGCCTGAATG C 21 18 base pairs nucleic acidsingle linear DNA NO NO unknown 3 GGACTGGGGT GAGGACGG 18 24 base pairsnucleic acid single linear DNA NO NO unknown 4 TTTCAGTAGA TAACGCACTGCTGG 24

What is claimed is:
 1. A method for reversing a tumorigenic state of acell comprising administering an agent that reduces the level oractivity of a DNA methyltransferase or increases the level or activityof a DNA dimethylase thereby reducing methylation of cytosine in a CpGdinucleotide in the cell, thereby correcting an aberrant methylationpattern in the DNA of the cell.
 2. The method of claim 1, wherein thereduction of the level of methylated cytosine in a CpG dinudeotide iseffected by inhibiting DNA methyltransferase activity.
 3. The method ofclaim 1, wherein the reduction of the level of methylated cytosine in aCpG dinucleotide is effected by increasing demethylase activity.